Effects of clenbuterol administration on mitochondrial morphology and its regulatory proteins in rat skeletal muscle

Abstract Clenbuterol induces a slow‐to‐fast fiber type transition in skeletal muscle. This muscle fiber transition decreased mitochondrial oxidative capacity and respiratory function. We hypothesized that the clenbuterol‐mediated reduction in oxidative capacity is associated with the alteration in mitochondrial morphology. To verify this hypothesis, we examined whether clenbuterol alters mitochondrial morphology and mitochondrial regulatory proteins in rat skeletal muscle. Clenbuterol was administered to rats via drinking water (30 mg/L) for 3 weeks. Myosin heavy chain (MHC) isoform composition, mitochondrial morphology, and fusion and fission regulatory protein levels in deep region and superficial region in tibialis anterior (TA) muscles were assessed. Clenbuterol induced the fiber type transition from slow to fast in both the regions of TA. The levels of optic atrophy protein 1, mitofusin 2, and mitochondrial fission 1, but not of dynamin‐related protein 1, significantly decreased in deep and superficial muscles after clenbuterol administration (P < 0.01). Also, observation using the transmission electron microscopy showed a decrease in mitochondrial volume (P < 0.05) and an increase in proportion of continuous or interacting mitochondria across Z‐lines (P < 0.05). We showed that clenbuterol administration induces a transition in the muscle fiber type composition toward fast phenotype and causes alterations in mitochondrial morphology with a concomitant decrease in mitochondrial fusion and fission regulatory protein levels. These mitochondrial morphological alterations may influence deleterious effects on skeletal muscle metabolism.


Introduction
Clenbuterol, a b2-adrenergic agonist, is commonly prescribed as a bronchodilator for the treatment of asthmatic patients. In addition to its anti-asthma effect, it causes a significant increase in muscle mass and strength in rodents when administered for long term (Zeman et al., 1988;Dodd et al., 1996). Owing to this anabolic effect, clenbuterol has the potential to ameliorate muscle wasting in pathological conditions (Lynch and Ryall, 2008), and also to be exploited by athletes.
Long-term clenbuterol administration induces a transition from slow-to-fast muscle fiber type composition, which is defined by myosin ATPase activity (Zeman et al., 1988) and myosin heavy chain (MHC) isoforms (Dodd et al., 1996;Kitaura et al., 2001;Oishi et al., 2002;Ohnuki et al., 2016); this transition is accompanied by increased muscle force and decreased fatigue tolerance (Zeman et al., 1988;Dodd et al., 1996). Muscle metabolic characteristics also change with the transition in the fiber type composition toward fast phenotype (Torgan et al., 1993;Dodd et al., 1996;Hoshino et al., 2012). We have previously reported that the levels of markers of mitochondrial content and respiratory function decrease with clenbuterol administration, indicating its deleterious effects on skeletal muscle metabolism (Hoshino et al., 2012).
Mitochondria are organized in a morphologically plastic network regulated by fusion and fission; these processes, termed mitochondrial dynamics, play a crucial role in the maintenance of functional mitochondria. The expression of mitochondrial dynamics proteins correlates with the oxidative capacity of muscle fibers (Iqbal et al., 2013). Recent evidence has shown that endurance exercise training leads to a more fused, elongated mitochondrial network, with muscle fiber type transition toward slow phenotype (Iqbal et al., 2013;Axelrod et al., 2018). However, it remains unclear whether clenbuterol, which induces slow-to-fast muscle fiber type transition, can alter mitochondrial morphology and the expression of dynamics regulatory proteins. We hypothesized that the clenbuterol-mediated reduction in oxidative capacity is not only attributable to muscle mitochondrial content reduction but also associated with concurrent changes in mitochondrial morphology. In this study, we first assessed proteins involved in oxidative phosphorylation (OXPHOS) as markers of mitochondrial content. Next, we examined whether clenbuterol administration affects protein levels involved in mitochondrial fusion and fission events. Furthermore, transmission electron microscopy analysis allowed us to assess changes in mitochondrial morphology in response to clenbuterol administration.

Animals and clenbuterol administration
We purchased 12 Wistar male rats (aged 8 weeks) from Japan SLC Inc. (Shizuoka, Japan). The rats were housed on a 12:12-h light-dark cycle in an air-conditioned room. Following a 3-to 7-day acclimation period, the animals were randomly divided into two treatment groups: control group (n = 6), which was provided with standard chow and water ad libitum for 3 weeks, and clenbuterol group (n = 6), which was provided with standard chow and treated for 3 weeks with clenbuterol (Sigma-Aldrich, St. Louis, MO, USA) administered via drinking water (30 mg/L), as previously reported (Stevens et al., 2000;Oishi et al., 2002;Hoshino et al., 2012). Two rats were housed in a cage (22 cm 9 38 cm with 20 cm height). Body weight and amount of water intake were recorded 3 times a week. Water intake per two animals in a cage was not significantly different between the two groups during the experimental period. After 3 weeks, the animals were anesthetized (65 mg of pentobarbital sodium/100 g body weight), and tibialis anterior (TA) muscles were excised. We separated into deep and superficial region for performing immunohistochemical staining and western blotting because we examined mitochondrial markers and mitochondrial respiratory function in these regions of TA in the previous study (Hoshino et al., 2012). Separation of the deep and superficial TA compartments was based on their distinct anatomical locations in the muscle of control animals. This separation is a common method to examine the effects of pharmacological and physiological stimulus on metabolic adaptation in different fiber type of rat skeletal muscles (Bonen et al., 2000;Enoki et al., 2006;Benton et al., 2008). Muscle samples were stored at À80°C until further immunohistochemical and western blot analyses. The treatment procedures were approved by the Institutional Animal Care and Use Committee of University of Electro-Communications (Tokyo, Japan; Approval No. 29).

Immunohistochemical staining
The fiber type composition of MHCI, IIa, and IIb was determined through immunohistochemical staining as described previously (Watanabe et al., 2017). Briefly, the excised TA muscle blocks were rapidly frozen in isopentane and cooled in liquid nitrogen. Serial 10-lm sections were made with a cryostat (CM1950; Leica Biosystems, Jena, Germany) at À20°C and mounted on polylysinecoated slides. MHC monoclonal antibodies obtained from Developmental Studies Hybridoma Bank (Iowa city, IA, USA) were used (dilution, 1:100; BA-F8 for MHCI, SC-71 for IIa, BF-F3 for IIb). The sections were allowed to be warmed at room temperature and were incubated in phosphate-buffered saline (PBS) (pH 7.5) at 25°C before further incubation with a primary antibody in a humidified box overnight at 4°C. Vectastain ABC kit (Burlingame, CA, USA) was used according to manufacturer's instructions to reveal the immunohistochemical reaction. Muscle fibers stained by each MHC isoform were counted within total 100 muscle fibers. The images were subsequently analyzed using ImageJ software.

Transmission electron microscopy
Muscle bundles (~10 fibers) were fixed with 2% glutaraldehyde and 2.5% formaldehyde in 0.1 M phosphate buffer (PB) solution (pH 7.4) for 24 h at 4°C and subsequently washed in 0.1 M PB for 12 h at 4°C. Thereafter, the bundles were post-fixed with 1% osmium tetroxide in 0.1 M PB for 2 h at 4°C with gentle shaking and then dehydrated through a graded series of ethanol at 25°C. After dehydration, the bundles were infiltrated with a graded mixture of propylene oxide and resin at 25°C and then embedded in longitudinal orientation on 100% resin at 60°C for 48 h. Afterward, 1-lm thick sections were made to check the orientation of the embedded bundle and section quality. Ultrathin sections were cut using a diamond knife on an ultramicrotome, and the sections were mounted on Pioloform filmed copper grids. After staining with uranyl acetate and lead citrate, the sections were photographed under a transmission electron microscope (TEM) using a charge-coupled device camera. From each fiber, 3-5 images were obtained at 930,700 magnification from central regions of the myofibrillar space.

Stereological methods
Mitochondrial volume was estimated using a standard stereological method (i.e., counting intersections) reported by Nielsen et al. (2010). The mitochondrial volume density (V v ) was estimated using the following formula: V v = A A , where A A denotes the mitochondrial area estimated as follows: 420 9 420-nm grids were superimposed on each TEM image, and then, the number of points that touched mitochondria were tallied and divided by total number of points on the grid (i.e., 462 points in this study). In addition, 4-6 images were taken per fiber, and the images were then analyzed by three investigators. The coefficients of error of mitochondrial volume were as follows: 0.17 (deep region of TA in the control group), 0.09 (superficial region of TA in the control group), 0.11 (deep region of TA in the clenbuterol group), and 0.09 (superficial region of the TA in clenbuterol group).

Mitochondrial membrane interactions
Intracellular mitochondria interact with neighboring mitochondria, and their reticulum constantly undergoes fusion and fission. Typically, in the longitudinal section, a pair of mitochondria can be observed on both sides of the Z-line (e.g., arrows in Fig. 3A). However, some pairs of mitochondria interact across Z-line, or a single mitochondrion spans Z-line. Using TEM micrographs, the number of Z-lines possessing mitochondria on both sides (Z-line total ) and the number of Z-lines where a pair of mitochondria spanned (Z-line spanned ) were counted. The proportion of Z-lines spanned by a continuous mitochondrion or interacting mitochondria (Z-line spanned /Z-line total) in each fiber was measured as reported by Picard et al. (2013). The coefficients of error of Z-linespanned /Z-line total were as follows: 0.13 (deep region of TA in the control group), 0.09 (superficial region of TA in the control group), 0.10 (deep region of TA in the clenbuterol group), and 0.06 (superficial region of TA in the clenbuterol group). Eventually, 431 and 632 pairs of mitochondria in superficial and deep TA, respectively, were analyzed.

Statistical analysis
All data were presented as mean AE standard error of the mean. Two-way repeated measures analysis of variance (ANOVA) was used to analyze the differences for the body weights. A two-way ANOVA was also used to analyze the between-group differences (control vs. clenbuterol) for muscles (deep vs. superficial). Besides, post hoc comparisons were performed using the Sidak procedure. All statistical analyses were performed using Graph-Pad Prism version 8.0 Software (GraphPad, San Diego, CA, USA). P-value < 0.05 was considered significant. 0.482 AE 0.01 g, clenbuterol: 0.585 AE 0.01 g) and relative muscle weight (control: 1.84 AE 0.04 mg/g body weight, clenbuterol: 2.24 AE 0.04 mg/g body weight) was significantly higher in the clenbuterol group than in the control group (P < 0.05).

Muscle fiber type composition.
Muscle fiber composition of MHCI was significantly lower in the clenbuterol group than in the control group in deep region, whereas MHCI was not detected in superficial region of TA in either group (P < 0.05, Fig1A and 1). Muscle fiber composition of MHCIIa tended to be lower in the clenbuterol group (P = 0.055, Fig. 1A and 1), whereas that of MHCIIb was significantly higher in the clenbuterol group than in the control group in both deep and superficial regions (P < 0.01, Fig. 1A and 1).

Mitochondrial proteins
The levels of all mitochondrial complex (I-V) proteins were significantly lower in the clenbuterol group than in the control group in both deep and superficial muscles (P < 0.01, Fig. 2A-F). Additionally, Opa1, Mfn2, and Fis1 levels were significantly lower in the clenbuterol group than in the control group (P < 0.05, Fig. 3A-C and E) in both deep and superficial muscles, whereas Drp1 levels remained unaffected (P > 0.05, Fig. 3D).

Mitochondrial volume and morphology
The electron micrographs of in the deep and superficial region of TA showed organized fibers with mitochondria in the both groups (Fig. 4A). Mitochondrial volume was 0.077 AE 0.013 lm 3 /lm 3 fiber volume (deep region of TA in the control group), 0.051 AE 0.005 lm 3 /lm 3 fiber volume (deep region of TA in the clenbuterol group), 0.030 AE 0.003 lm 3 /lm 3 fiber volume (superficial region of TA in the control group), and 0.021 AE 0.002 lm 3 /lm 3 fiber volume (superficial region of TA in the clenbuterol group). Mitochondrial volume was significantly lower in the clenbuterol group than in the control group in both the regions (P < 0.05, Fig. 4A and 4). The proportions of continuous or interacting mitochondria across Z-lines (Zline spanned /Z-line total ) were 23.9% AE 3.2% in the deep region of TA in the control group and 43.1% AE 3.9% in the superficial region of TA in the control group; these values were consistent with previously reported values (Picard et al., 2013). The proportion of mitochondria spanning Z-line was significantly higher in the clenbuterol group than in the control group in the deep (49.4% AE 5.1%) and superficial (53.3% AE 3.3%) regions (P < 0.05, Fig. 4C). Furthermore, mitochondria in the clenbuterol group showed disrupted and abnormal mitochondrial cristae structure, which is classic ultrastructural signs of mitochondrial dysfunction (Fig. 4D).

Discussion
We examined whether clenbuterol alters mitochondrial morphology and mitochondrial protein levels in deep and superficial region of TA muscles. Along with the fiber type transition from slow to fast, we found that   clenbuterol decreased the levels of mitochondrial OXPHOS proteins as well as those of proteins involved in fusion (Mfn2, Opa1) and fission (Fis1). Furthermore, we observed a reduction in the mitochondrial volume and an increase in the proportion of continuous or interacting mitochondria across the Z-line. These results suggest that clenbuterol-induced slow-to-fast muscle fiber type transition alters mitochondrial dynamics protein and mitochondrial morphology.
The transition in fiber type composition toward fast phenotype was observed in both deep and superficial regions after clenbuterol administration over 3 weeks and was consistent with previous reports (Zeman et al., 1988;Dodd et al., 1996;Kitaura et al., 2001;Oishi et al., 2002;Ohnuki et al., 2016). Accompanied with the slow-to-fast fiber type transition, mitochondrial volume analyzed using TEM decreased in both deep and superficial regions following clenbuterol administration. . Effect of clenbuterol administration on mitochondrial morphology. Representative micrographs (A). Mitochondrial volume was estimated using standard stereological methods (C). Mitochondria can be found among the myofibrils, with the majority of them aligned with the Z-line. Some Z-lines possess mitochondria on both sides (i.e., yellow and red arrows); red arrows indicate continuous or interacting mitochondria across Z-lines; yellow arrows indicate Z-line possessing two mitochondria without any interaction. (B). Mitochondria in the clenbuterol group showed disrupted mitochondrial cristae structure (white arrows) (D). Values (mean AE standard error of mean) are average of 9-12 fibers obtained from four rats. *P < 0.05, significant effect on control group vs. clenbuterol group. † P < 0.05, significant effect on superficial region vs. deep region.